A determination of the relationship between the molecular basis of genetic alterations and phenotype generally utilizes accurate two- and three-dimensional characterization of microstructure of biological specimens. However, motion and small dimensions make many living biological specimens can be more difficult to evaluate.
Optical techniques offer the potential to image the biological specimens at a high resolution. For certain applications, optical imaging based on endogenous contrast can be advantageous over techniques that require exogenous agents, since such beneficial procedures can allow the analysis of the specimen in its native state and at multiple time points, with a small amount of preparation. As an example, several endogenous-contrast imaging modalities are described herein for visualizing embryonic heart microstructure: two exemplary forms of optical coherence tomography (“OCT”) as described in D. Huang et al., “Optical coherence tomography,” Science 254, pp. 1178-1181 (1991), time-domain optical coherence tomography (“TD-OCT”) as described in S. A. Boppart et al., “Investigation of developing embryonic morphology using optical coherence tomography,” Dev Biol 177, pp. 54-63 (1996), and optical frequency domain imaging (“OFDI”) as described in M. A. Choma et al., “Sensitivity advantage of swept source and Fourier domain optical coherence tomography,” Optics Express 11, pp. 2183-2189 (2003); and S. H. Yun et al., “High-speed optical frequency-domain imaging,” Optics Express 11, pp 2953-2963 (2003).
Additional examples can be provided and utilized including two reflectance microscopy techniques, e.g., full-field optical coherence microscopy (“FFOCM”) as described in E. Beaurepaire et al., “Full-field optical coherence microscopy,” Optics Letters 23, pp. 244-246 (1998); A. Dubois et al., “Ultrahigh-resolution full-field optical coherence tomography,” Appl Opt 43, pp. 2874-2883 (2004); and G. Moneron et al., “Stroboscopic ultrahigh-resolution full-field optical coherence tomography,” Opt Lett 30, pp. 1351-1353 (2005), and spectrally encoded confocal microscopy (“SECM”) as described in G. J. Tearney et al., “Spectrally encoded confocal microscopy,” Optics Letters 23, pp. 1152-1154 (1998); and C. Boudoux et al., “Rapid wavelength-swept spectrally encoded confocal microscopy,” Optics Express 13, pp. 8214-8221 (2005).
For example, the TDOCT techniques can use low-coherence interferometry to obtain cross-sectional images with ˜10 μm resolution and at depths of up to 2 mm. (See S. A. Boppart et al., “Noninvasive assessment of the developing Xenopus cardiovascular system using optical coherence tomography,” Proc Natl Acad Sci U S A 94, pp. 4256-4261 (1997); S. Yazdanfar et al., “High resolution imaging of in vivo cardiac dynamics using color Doppler optical coherence tomography,” Optics Express 1, pp. 424-431 (1997); T. M. Yelbuz et al., “Optical coherence tomography: a new high-resolution imaging technology to study cardiac development in chick embryos,” Circulation 106, pp. 2771-2774 (2002); V. X. D. Yang et al., “High speed, wide velocity dynamic range Doppler optical coherence tomography (Part II): Imaging in vivo cardiac dynamics of Xenopus laevis,” Optics Express 11, pp. 1650-1658 (2003); and W. Luo et al., “Three-dimensional optical coherence tomography of the embryonic murine cardiovascular system” Journal of biomedical optics 11, 021014 (2006).
The exemplary OFDI technique can be considered as a derivative of the TDOCT techniques that may enable an acquisition of images at significantly higher frame rates as described in R. Huber et al., “Three-dimensional and C-mode OCT imaging with a compact, frequency swept laser source at 1300 nm,” Optics Express 13, pp. 10523-10538 (2005). The high speed of the OFDI techniques can enable an implementation of a true four-dimensional (4D) microscopy (e.g., three-dimensional microscopy as a function of time). Full-field optical coherence microscopy (“FFOCM”) techniques can utilize low-coherence interferometry and higher numerical aperture objective lenses to attain resolution at the subcellular level in all three dimensions. Such FFOCM techniques are likely considerably slower than the OFDI techniques. The exemplary SECM techniques can have a form of the reflectance confocal microscopy using which it may be possible to obtain two-dimensional images with micron-level resolution, at significantly higher speeds than possibly obtained using the FFOCM techniques.
While each of these natural-contrast procedures can individually be used for imaging a microstructure of the embryonic heart, when combined, these procedures can provide a powerful set of tools for two-, three-, and four-dimensional characterization of early myocardial morphology and dynamics. A combination of these different modalities into one single microscopy device may have additional advantages such as, e.g., (a) a comparison of images in different formats, different resolutions, and fields of view, (b) a simultaneous acquisition of both structural and function information, and/or (c) these tasks can be accomplished using one instrument without requiring moving or altering the specimen.